We seek to understand the structural basis for negatively cooperative3 ligand binding by transferrin receptor (TfR). TfR is a membrane-bound homodimer that binds two ligands: iron loaded transferrin (Fe-Tf) and HFE, the protein mutated in the iron overload disease hereditary. hemochromatosis. Binding of HFE to one TfR chain lowers the affinity for binding Fe-Tf or another HFE to the other chain. We solved crystal structures of HFE alone and a 2:2 TfR/HFE complex in which one HFE is bound to each chain of the TfR homodimer. The TfR/HFE structure reveals that HFE binding induces changes at the TfR dimer interface, suggesting that binding to one polypeptide chain of the TfR dimer transmits structural changes to the other TfR chain that influence binding of Fe-Tf or another HFE. To elucidate how structural changes influencing binding are transmitted, we will characterize the biochemical and structural properties of a 2:1 TfR/HFE complex (TfR dimer binding only one HFE) and a Fe-TfR/HFE ternary complex. These complexes will be prepared using a heterodimeric version of TfR in which mutations have been introduced such that one polypeptide chain can bind HFE but not Fe-Tf, and the other polypeptide chain can bind Fe-Tf but not HFE. We will first identify residues that eliminate binding of HFE or Fe-Tf by measuring the affinities of site-directed TfR mutants for Fe-Tf or HFE using a quantitative biosensor assay. Since HFE and Fe-Tf bind to an overlapping site on TfR, residues within or near the HFE binding site on TFR (which is known from the TfR/HFE crystal structure) should form part of the Fe-Tf binding site. Upon identification of TfR mutants that retain binding for one ligand, but not the other, we will produce TfR heterodimers by expressing both mutant TfR chains in the same cell. TfR heterodimers will be purified from homodimers using different affinity tags attached to each TfR chain. Heterodimeric TfR will be used to solve crystal structures of a 2:1 TfR/HFE complex and a 1:2:1 Fe-Tf/TfR/HFE complex. Comparisons with the structures of TfR alone and the 2:2 TfR/HFE complex will elucidate the structural effects of ligand binding to one side of the TfR dimer. Other TfR mutants will be analyzed to elucidate the structural mechanism for TfR's sharply pH dependent binding behavior. TfR binds apo-Tf at the acidic pH of endosomes (greater than or equal to pH 6.5), but not at the slightly basic pH of the cell surface (pH 7.4) and binds to HFE with the opposite pH dependence. Results from these studies will be critical for understanding how interactions between TfR, HFE, and Fe-Tf are involved in regulating iron homeostasis.